Cell-type specificity in the biotrophic interaction of Ustilago maydis and its host plant maize

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The biotrophic fungus Ustilago maydis infects all aerial parts of its host plant maize, where it causes the formation of large plant tumors. This project builds on our initial hypothesis that U. maydis deploys organ-specific sets of secreted effector proteins to cause tumors in the different host tissues. During the previous project phase we verified this idea and identified a set of organ-specific effectors, which are required for formation of tumors in maize leaves, but not in the inflorescences. One of these effectors, See1, was found to be required for the induction of DNA synthesis in maize leaves, which is re-activated by the fungus at the onset of tumor formation. Detailed cytological analyses of infected maize leaves revealed that U. maydis induced leaf tumors are formed by two distinct cellular processes. The bundle sheath cells are the origin of hyperplasic tumor cells (HPTs), while the mesophyll is transformed into hypertrophic tumor cells (HTTs). Strikingly, the See1 effector acts as an essential inducer of the cell cycle reactivation specifically in the bundle sheath cells. Laser-capture microdissection was used to isolate RNA for transcriptome analysis from HTTs and HPTs, as well as HPTs induced by the see1 deletion mutant. This approach revealed cell-type specific gene expression of U. maydis effector gene. Moreover, it provided a set of nine candidates for hypertrophy-related effectors. A comparative transcriptome analysis of U. maydis with the closely related pathogen Sporisorium reilianum, which cannot induce leaf tumors, revealed differential transcriptional regulation being particularly relevant for the function of leaf- and cell-type specific effectors.Based on these results, this project aims to understand the cell-type specific mechanisms underlying U. maydis induced tumor formation. After having identified See1 as an effector being required for the induction of plant cell proliferation, we now aim to dissect how U. maydis induces hypertrophy. To this end, we will identify and functionally characterize the hypertrophy-inducing effector(s) of U. maydis. We will identify the plant targets of these effector(s) to understand the molecular basis of hypertrophy induction by U. maydis. Finally, we proposed a novel approach to functionally analyze hypertrophy-/tumor-inducing effectors by a CRISPR-Cas9 mediated gene replacement approach in S. reilianum.We expect this project to provide novel insights into the pathogen-triggered induction of hypertrophy by fungal effectors. Ultimately, our strategy aims to define a functional effector-set able to reconstruct tumor formation in plants, which will help us to understand this exciting process on the mechanistic level.